During the 9 years of its existence, the AHDL has worked to solidify and extend observations that uncovered a previously unappreciated aspect of innate immunity: the requirement that a potent microbial stimulus, lipopolysaccharide (LPS), be inactivated by a host enzyme before homeostasis can be restored following Gram-negative bacterial diseases. The enzyme, acyloxyacyl hydrolase (AOAH), has been the focus of our lab's work for many years. We have carried out and/or completed the following studies during these years: a. What happens to LPS in vivo? When LPS enters the bloodstream, a large fraction of it binds rapidly to circulating lipoproteins, inserting in the lipoprotein particle in such a way that the bioactive (stimulatory) LPS moiety is buried and thus inactive. Lipoprotein-bound LPS is largely taken up by the liver. We found that, contrary to published reports and our own expectations, the LPS in high density lipoprotein (HDL) particles is mainly taken up by Kupffer cells, not hepatocytes, and that it undergoes deacylation/inactivation by AOAH. This again shows the central role played by AOAH in disposing of bioactive LPS in vivo. b. What is the basis for prolonged macrophage tolerance (reprogramming) following exposure to LPS in vivo? Aoah-/- mice, when exposed to small amounts of LPS, develop long-lasting hyporesponsiveness to a second LPS exposure. To understand the molecular basis for this phenomenon, which resembles (and may be a useful model for) the state of immunosuppression that occurs in humans following bacterial infections, we have focused on the peritoneal macrophage. An extensive series of transfer studies (involving the transfer of peritoneal macrophages from mice with one phenotype/genotype into recipient mice of a different phenotype/genotype) have shown us that the trigger for prolonged reprogramming in Aoah-/- macrophages is not, as expected, a fixed change in the cells, but rather the presence of fully active extracellular LPS within the peritoneal cavity. We are now looking for non-LPS substrates for AOAH. Candidates include oxidized phospholipids, bacterial lipopeptides, and 2-arachidonoylglycerol. These studies seem promising.